Dissection of a CTCF topological boundary uncovers principles of enhancer-oncogene regulation.

Enhancer-gene communication is dependent on topologically associating domains (TADs) and boundaries enforced by the CCCTC-binding factor (CTCF) insulator, but the underlying structures and mechanisms remain controversial. Here, we investigate a boundary that typically insulates fibroblast growth factor (FGF) oncogenes but is disrupted by DNA hypermethylation in gastrointestinal stromal tumors (GISTs). The boundary contains an array of CTCF sites that enforce adjacent TADs, one containing FGF genes and the other containing ANO1 and its putative enhancers, which are specifically active in GIST and its likely cell of origin. We show that coordinate disruption of four CTCF motifs in the boundary fuses the adjacent TADs, allows the ANO1 enhancer to contact FGF3, and causes its robust induction. High-resolution micro-C maps reveal specific contact between transcription initiation sites in the ANO1 enhancer and FGF3 promoter that quantitatively scales with FGF3 induction such that modest changes in contact frequency result in strong changes in expression, consistent with a causal relationship.

Modeling epigenetic lesions that cause gliomas.

Epigenetic lesions that disrupt regulatory elements represent potential cancer drivers. However, we lack experimental models for validating their tumorigenic impact. Here, we model aberrations arising in isocitrate dehydrogenase-mutant gliomas, which exhibit DNA hypermethylation. We focus on a CTCF insulator near the PDGFRA oncogene that is recurrently disrupted by methylation in these tumors. We demonstrate that disruption of the syntenic insulator in mouse oligodendrocyte progenitor cells (OPCs) allows an OPC-specific enhancer to contact and induce Pdgfra, thereby increasing proliferation. We show that a second lesion, methylation-dependent silencing of the Cdkn2a tumor suppressor, cooperates with insulator loss in OPCs. Coordinate inactivation of the Pdgfra insulator and Cdkn2a drives gliomagenesis in vivo. Despite locus synteny, the insulator is CpG-rich only in humans, a feature that may confer human glioma risk but complicates mouse modeling. Our study demonstrates the capacity of recurrent epigenetic lesions to drive OPC proliferation in vitro and gliomagenesis in vivo.

Systematic detection of m6A-modified transcripts at single-molecule and single-cell resolution.

Epigenetic modifications control the stability and translation of mRNA molecules. Here, we present a microscopy-based platform for quantifying modified RNA molecules and for relating the modification patterns to single-cell phenotypes. We directly capture mRNAs from cell lysates on oligo-dT-coated coverslips, then visually detect and sequence individual m6A-immunolabled transcripts without amplification. Integration of a nanoscale device enabled us to isolate single cells on the platform, and thereby relate single-cell m6A modification states to gene expression signatures and cell surface markers. Application of the platform to MUTZ3 leukemia cells revealed a marked reduction in cellular m6A levels as CD34+ leukemic progenitors differentiate to CD14+ myeloid cells. We then coupled single-molecule m6A detection with fluorescence in situ hybridization (FISH) to relate mRNA and m6A levels of individual genes to single-cell phenotypes. This single-cell multi-modal assay suite can empower investigations of RNA modifications in rare populations and single cells.

Activity of immunoproteasome inhibitor ONX-0914 in acute lymphoblastic leukemia expressing MLL-AF4 fusion protein.

Proteasome inhibitors bortezomib and carfilzomib are approved for the treatment of multiple myeloma and mantle cell lymphoma and have demonstrated clinical efficacy for the treatment of acute lymphoblastic leukemia (ALL). The t(4;11)(q21;q23) chromosomal translocation that leads to the expression of MLL-AF4 fusion protein and confers a poor prognosis, is the major cause of infant ALL. This translocation sensitizes tumor cells to proteasome inhibitors, but toxicities of bortezomib and carfilzomib may limit their use in pediatric patients. Many of these toxicities are caused by on-target inhibition of proteasomes in non-lymphoid tissues (e.g., heart muscle, gut, testicles). We found that MLL-AF4 cells express high levels of lymphoid tissue-specific immunoproteasomes and are sensitive to pharmacologically relevant concentrations of specific immunoproteasome inhibitor ONX-0914, even in the presence of stromal cells. Inhibition of multiple active sites of the immunoproteasomes was required to achieve cytotoxicity against ALL. ONX-0914, an inhibitor of LMP7 (ß5i) and LMP2 (ß1i) sites of the immunoproteasome, and LU-102, inhibitor of proteasome ß2 sites, exhibited synergistic cytotoxicity. Treatment with ONX-0914 significantly delayed the growth of orthotopic ALL xenograft tumors in mice. T-cell ALL lines were also sensitive to pharmacologically relevant concentrations of ONX-0914. This study provides a strong rationale for testing clinical stage immunoproteasome inhibitors KZ-616 and M3258 in ALL.

Parallel Single-Cell RNA-Seq and Genetic Recording Reveals Lineage Decisions in Developing Embryoid Bodies.

Early developmental specification can be modeled by differentiating embryonic stem cells (ESCs) to embryoid bodies (EBs), a heterogeneous mixture of three germ layers. Here, we combine single-cell transcriptomics and genetic recording to characterize EB differentiation. We map transcriptional states along a time course and model cell fate trajectories and branchpoints as cells progress to distinct germ layers. To validate this inferential model, we propose an innovative inducible genetic recording technique that leverages recombination to generate cell-specific, timestamp barcodes in a narrow temporal window. We validate trajectory architecture and key branchpoints, including early specification of a primordial germ cell (PGC)-like lineage from preimplantation epiblast-like cells. We further identify a temporally defined role of DNA methylation in this PGC-epiblast decision. Our study provides a high-resolution lineage map for an organoid model of embryogenesis, insights into epigenetic determinants of fate specification, and a strategy for lineage mapping of rapid differentiation processes.

An Integrative Model of Cellular States, Plasticity, and Genetics for Glioblastoma.

Diverse genetic, epigenetic, and developmental programs drive glioblastoma, an incurable and poorly understood tumor, but their precise characterization remains challenging. Here, we use an integrative approach spanning single-cell RNA-sequencing of 28 tumors, bulk genetic and expression analysis of 401 specimens from the The Cancer Genome Atlas (TCGA), functional approaches, and single-cell lineage tracing to derive a unified model of cellular states and genetic diversity in glioblastoma. We find that malignant cells in glioblastoma exist in four main cellular states that recapitulate distinct neural cell types, are influenced by the tumor microenvironment, and exhibit plasticity. The relative frequency of cells in each state varies between glioblastoma samples and is influenced by copy number amplifications of the CDK4, EGFR, and PDGFRA loci and by mutations in the NF1 locus, which each favor a defined state. Our work provides a blueprint for glioblastoma, integrating the malignant cell programs, their plasticity, and their modulation by genetic drivers.

GABPß1L Wakes Up TERT.

TERT catalyzes telomere maintenance. While silenced in most normal somatic cells, TERT is expressed in cancer, often due to promoter mutations, facilitating replicative immortality. In this issue of Cancer Cell, Mancini et al. demonstrate that GABPß1L is required for mutant TERT promoter activity, thus identifying a potential therapeutic target.

A recombinant lentiviral PDGF-driven mouse model of proneural glioblastoma.

Background: Mouse models of glioblastoma (GBM), the most aggressive primary brain tumor, are critical for understanding GBM pathology and can contribute to the preclinical evaluation of therapeutic agents. Platelet-derived growth factor (PDGF) signaling has been implicated in the development and pathogenesis of GBM, specifically the proneural subtype. Although multiple mouse models of PDGF-driven glioma have been described, they require transgenic mice engineered to activate PDGF signaling and/or impair tumor suppressor genes and typically represent lower-grade glioma. Methods: We designed recombinant lentiviruses expressing both PDGFB and a short hairpin RNA targeting Cdkn2a to induce gliomagenesis following stereotactic injection into the dentate gyrus of adult immunocompetent mice. We engineered these viruses to coexpress CreERT2 with PDGFB, allowing for deletion of floxed genes specifically in transduced cells, and designed another version of this recombinant lentivirus in which enhanced green fluorescent protein was coexpressed with PDGFB and CreERT2 to visualize transduced cells. Results: The dentate gyrus of injected mice showed hypercellularity one week post-injection and subsequently developed bona fide tumors with the pathologic hallmarks of GBM leading to a median survival of 77 days post-injection. Transcriptomic analysis of these tumors revealed a proneural gene expression signature. Conclusion: Informed by the genetic alterations observed in human GBM, we engineered a novel mouse model of proneural GBM. While reflecting many of the advantages of transgenic mice, this model allows for the facile in vivo testing of gene function in tumor cells and makes possible the rapid production of large numbers of immunocompetent tumor-bearing mice for preclinical testing of therapeutics.

MiR-338-3p regulates neuronal maturation and suppresses glioblastoma proliferation.

Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral "sponge" to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.

ID2 promotes survival of glioblastoma cells during metabolic stress by regulating mitochondrial function.

Tumor cells proliferate in cellular environments characterized by a lack of optimal tissue organization resulting oftentimes in compromised cellular metabolism affecting nutrition, respiration, and energetics. The response of tumor cells to adverse environmental conditions is a key feature affecting their pathogenicity. We found that inhibitor of DNA binding 2 (ID2) expression levels significantly correlate with the ability of glioblastoma (GBM)-derived cell lines to survive glucose deprivation. ID2 suppressed mitochondrial oxidative respiration and mitochondrial ATP production by regulating the function of mitochondrial electron transport chain (mETC) complexes, resulting in reduced superoxide and reactive oxygen species (ROS) production from mitochondria. ID2 suppression of ROS production reduced mitochondrial damage and enhanced tumor cell survival during glucose deprivation. Bioinformatics analysis of GBM gene expression data from The Cancer Genome Atlas (TCGA) database revealed that expression of ID2 mRNA is unique among ID gene family members in correlating with the expression of nuclear genes involved in mitochondrial energy metabolism and assembly of mETC. Our data indicate that the expression level of ID2 in GBM cells can predict the sensitivity of GBM-derived tumor cells to decreased glucose levels. Low levels of ID2 expression in human GBM tissues may identify a clinical group in which metabolic targeting of glycolytic pathways can be expected to have the greatest therapeutic efficacy.

Immune modulation associated with vascular endothelial growth factor (VEGF) blockade in patients with glioblastoma.

BACKGROUND: Vascular endothelial growth factor (VEGF), in addition to being pro-angiogenic, is an immunomodulatory cytokine systemically and in the tumor microenvironment. We previously reported the immunomodulatory effects of radiation and temozolomide (TMZ) in newly diagnosed glioblastoma. This study aimed to assess changes in peripheral blood mononuclear cell (PBMC) populations, plasma cytokines, and growth factor concentrations following treatment with radiation, TMZ, and bevacizumab (BEV). METHODS: Eleven patients with newly diagnosed glioblastoma were treated with radiation, TMZ, and BEV, following surgery. We measured immune-related PBMC subsets using multi-parameter flow cytometry and plasma cytokine and growth factor concentrations using electrochemiluminescence-based multiplex analysis at baseline and after 6 weeks of treatment. RESULTS: The absolute number of peripheral blood regulatory T cells (Tregs) decreased significantly following treatment. The lower number of peripheral Tregs was associated with a CD4+ lymphopenia, and thus, the ratio of Tregs to PBMCs was unchanged. The addition of bevacizumab to standard radiation and temozolomide led to the decrease in the number of circulating Tregs when compared with our prior study. There was a significant decrease in CD8+ cytotoxic and CD4+ recent thymic emigrant T cells, but no change in the number of myeloid-derived suppressor cells. Significant increases in plasma VEGF and placental growth factor (PlGF) concentrations were observed. CONCLUSIONS: Treatment with radiation, TMZ, and BEV decreased the number but not the proportion of peripheral Tregs and increased the concentration of circulating VEGF. This shift in the peripheral immune cell profile may modulate the tumor environment and have implications for combining immunotherapy with anti-angiogenic therapy.

PDGF Engages an E2F-USP1 Signaling Pathway to Support ID2-Mediated Survival of Proneural Glioma Cells.

Glioblastoma is the most aggressive primary brain tumor and responds poorly to currently available therapies. Transcriptomic characterization of glioblastoma has identified distinct molecular subtypes of glioblastoma. Gain-of-function alterations leading to enhanced platelet-derived growth factor (PDGF) signaling are commonly observed in the proneural subtype of glioblastoma and can drive gliomagenesis. However, little is known about the downstream effectors of PDGF signaling in glioblastoma. Using a mouse model of proneural glioma and comparative transcriptomics, we determined that PDGF signaling upregulated ubiquitin-specific peptidase 1 (Usp1) to promote the survival of murine proneural glioma cells. Mechanistically, we found that PDGF signaling regulated the expression of the E2F transcription factors, which directly bound to and activated Usp1 Furthermore, PDGF-mediated expression of USP1 led to the stabilization of Inhibitor of DNA-binding 2 (ID2), which we found to be required for glioma cell survival. Genetic ablation of Id2 delayed tumor-induced mortality, and pharmacologic inhibition of USP1, resulting in decreased ID2 levels, also delayed tumorigenesis in mice. Notably, decreased USP1 expression was associated with prolonged survival in patients with proneural glioblastoma, but not with other subtypes of glioblastoma. Collectively, our findings describe a signaling cascade downstream of PDGF that sustains proneural glioblastoma cells and suggest that inhibition of the PDGF-E2F-USP1-ID2 axis could serve as a therapeutic strategy for proneural glioblastoma featuring increased PDGF signaling. Cancer Res; 76(10); 2964-76. ©2016 AACR.

Pediatric Brain Tumors: Current Knowledge and Therapeutic Opportunities.

Great progress has been made in many areas of pediatric oncology. However, tumors of the central nervous system (CNS) remain a significant challenge. A recent explosion of data has led to an opportunity to understand better the molecular basis of these diseases and is already providing a foundation for the pursuit of rationally chosen therapeutics targeting relevant molecular pathways. The molecular biology of pediatric brain tumors is shifting from a singular focus on basic scientific discovery to a platform upon which insights are being translated into therapies.

Regulatory T cells are not a strong predictor of survival for patients with glioblastoma.

BACKGROUND: Regulatory T cells (Tregs) are potentially prognostic indicators in patients with glioblastoma. If differences in frequency of Tregs in tumor or blood account for substantial variation in patient survival, then reliably measuring Tregs may enhance treatment selection and improve outcomes. METHODS: We measured Tregs and CD3+ T cells in tumors and blood from 25 patients with newly diagnosed glioblastoma. Tumor-infiltrating Tregs and CD3+ T cells, measured by quantitative DNA demethylation analysis (epigenetic qPCR) and by immunohistochemistry, and peripheral blood Treg proportions measured by flow cytometry were correlated with patient survival. Additionally, we analyzed data from The Cancer Genome Atlas (TCGA) to correlate the expression of Treg markers with patient survival and glioblastoma subtypes. RESULTS: Tregs, as measured in tumor tissue and peripheral blood, did not correlate with patient survival. Although there was a correlation between tumor-infiltrating Tregs expression by epigenetic qPCR and immunohistochemistry, epigenetic qPCR was more sensitive and specific. Using data from TCGA, mRNA expression of Forkhead box protein 3 (FoxP3) and Helios and FoxP3 methylation level did not predict survival. While the classical glioblastoma subtype corresponded to lower expression of Treg markers, these markers did not predict survival in any of the glioblastoma subtypes. CONCLUSIONS: Although immunosuppression is a hallmark of glioblastoma, Tregs as measured in tissue by gene expression, immunohistochemistry, or demethylation and Tregs in peripheral blood measured by flow cytometry do not predict survival of patients. Quantitative DNA demethylation analysis provides an objective, sensitive, and specific way of identifying Tregs and CD3+ T cells in glioblastoma.

Serine/threonine kinase 17A is a novel candidate for therapeutic targeting in glioblastoma.

STK17A is a relatively uncharacterized member of the death-associated protein family of serine/threonine kinases which have previously been associated with cell death and apoptosis. Our prior work established that STK17A is a novel p53 target gene that is induced by a variety of DNA damaging agents in a p53-dependent manner. In this study we have uncovered an additional, unanticipated role for STK17A as a candidate promoter of cell proliferation and survival in glioblastoma (GBM). Unexpectedly, it was found that STK17A is highly overexpressed in a grade-dependent manner in gliomas compared to normal brain and other cancer cell types with the highest level of expression in GBM. Knockdown of STK17A in GBM cells results in a dramatic alteration in cell shape that is associated with decreased proliferation, clonogenicity, migration, invasion and anchorage independent colony formation. STK17A knockdown also sensitizes GBM cells to genotoxic stress. STK17A overexpression is associated with a significant survival disadvantage among patients with glioma which is independent of age, molecular phenotype, IDH1 mutation, PTEN loss, and alterations in the p53 pathway and partially independent of grade. In summary, we demonstrate that STK17A provides a proliferative and survival advantage to GBM cells and is a potential target to be exploited therapeutically in patients with glioma.

Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: role of ß-catenin/connexin43 association.

Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of ß-catenin, α-catenin and ZO-2, it decreased nuclear levels of ß-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, ß-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, ß-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering ß-catenin away from nucleus.

Gap junctions mediate STAT5-independent ß-casein expression in CID-9 mammary epithelial cells.

Crosstalk between gap junction intracellular communication (GJIC), STAT5 and OCT-1 in gap junction (GJ)-dependent ß-casein expression was investigated. CID-9 mammary cells plated with prolactin on non-adherent substratum (poly-HEMA) expressed ß-casein independent of STAT5 only in the presence of the GJIC inducer, cAMP. Nuclear STAT5 levels were not detectable. By contrast, cells on EHS-drip expressed ß-casein in a STAT5-dependent manner and nuclear STAT5 levels were up-regulated. A 75 kDa OCT-1 isoform was detected in conditions that induced ß-casein expression regardless of substratum. Interestingly, 40 and 28 kDa OCT-1 isoforms were induced in cells on polyHEMA with cAMP. Electrophoretic mobility shift assays (EMSA) for OCT-1 revealed two band shifts in cells on polyHEMA with cAMP and on EHS-drip, which were repressed by the GJIC inhibitor, 18α-GA. These studies demonstrated that mammary cells on polyHEMA expressed ß-casein in response to prolactin in a pathway that involves GJIC and OCT-1 and is independent of STAT5 nuclear translocation.

Splice variant-specific cellular function of the formin INF2 in maintenance of Golgi architecture.

INF2 is a unique formin that can both polymerize and depolymerize actin filaments. Mutations in INF2 cause the kidney disease focal and segmental glomerulosclerosis. INF2 can be expressed as two C-terminal splice variants: CAAX and non-CAAX. The CAAX isoform contains a C-terminal prenyl group and is tightly bound to endoplasmic reticulum (ER). The localization pattern and cellular function of the non-CAAX isoform have not been studied. Here we find that the two isoforms are expressed in a cell type-dependent manner, with CAAX predominant in 3T3 fibroblasts and non-CAAX predominant in U2OS, HeLa, and Jurkat cells. Although INF2-CAAX is ER localized in an actin-independent manner, INF2-non-CAAX localizes in an actin-dependent meshwork pattern distinct from ER. INF2-non-CAAX is loosely attached to this meshwork, being extracted by brief digitonin treatment. Suppression of INF2-non-CAAX causes fragmentation of the Golgi apparatus. This effect is counteracted by treatment with the actin monomer-sequestering drug latrunculin B. We also find discrete patches of actin filaments in the peri-Golgi region, and these patches are reduced upon INF2 suppression. Our results suggest that the non-CAAX isoform of INF2 serves a distinct cellular function from that of the CAAX isoform.